Superantigens (SAGs) are a class of pyrogenic bacterial toxins that elicit massive T cell activation through simultaneous binding to MHC class II molecules and T cell receptors. This activation results in the release of massive amounts of inflammatory cytokines, ultimately causing toxic shock syndrome. Due to their extreme virulence and ease of dissemination, SAGs have been identified as CDC Category B Agents of Bioterrorism. The development of therapeutics to treat SAG-mediated disease, against which no drug or vaccine currently exists, is therefore an important component of national defense against biological weapons. The US military has long considered the SAG staphylococcal enterotoxin B (SEB) as a major incapacitating and lethal threat. However, SEB is only one of 22 SAGs produced by Staphylococcus aureus and Streptococcus pyogenes, each capable of inducing toxic shock, some at far lower doses than SEB. Therefore, any credible program to develop therapeutics for SAG-induced disease must include a comprehensive strategy for neutralizing a broad spectrum of toxins, not simply SEB. We propose to use directed evolution methods (yeast display) to develop high-affinity protein antagonists of staphylococcal and streptococcal SAGs that will specifically abrogate their interaction with cell surface receptors in vivo, thereby blocking the pyrogenic and lethal cascades. Since SAGs possess highly conserved MHC-binding sites, soluble MHC class II molecules engineered to bind SAGs with high affinity should effectively neutralize a broad spectrum of SAGs. To develop such therapeutics, we will: 1) Use yeast display technology to generate soluble human HLA-DR molecules with nanomolar affinity for SAGs. 2) Characterize the interaction of affinity-matured HLA-DR molecules with SAGs, both biophysically and structurally. 3) Test the ability of soluble forms of high-affinity HLA-DR molecules to inhibit SAG activity in vitro using T cell stimulation assays. 4) Test the ability of high-affinity HLA-DR molecules to prevent toxicity in mouse and rabbit models of SAG-induced toxic shock. 5) Engineer improved forms (e.g. tetramers) of the antagonists discovered in Aims 1-4 in order to increase avidity and prolong serum lifetime. We expect that the strategies developed here for bacterial SAGs will be directly applicable to the development of antagonists against other toxins considered potential agents of bioterrorism. Superantigens are bacterial toxins that have been identified by the US Centers for Disease Control and Prevention as potential agents of bioterrorism. Superantigens cause toxic shock syndrome, an acute onset illness characterized by hypotension and multiple organ failure, leading to death. The goal of this proposal is to use protein engineering to develop novel therapeutics for neutralizing the lethal effects of bacterial superantigens. [unreadable] [unreadable] [unreadable]